We have analyzed a variety of molecules obtained from various sources to get the quantitative information. Additionally, we are developing methods to improve the quantitative information that can be gained. 1. Method Development. The use of label-free approaches for quantitative proteomics studies has been implemented. This methodology involves data-independent analyses (MSe) and an online UPLC method suitable for the analysis of proteins from complex samples. In theory, MSe can rapidly acquire accurate mass precursor and fragment ion information while simultaneously obtaining fairly accurate quantitative profiles from every detectable component in the sample. Additionally, we are incorporating ion mobility methods in an effort to gain further information. Ion mobility is a useful tool to aid in mass spectrometry applications because it allows for the measurement of the collisional cross section of a molecule and gives information about the three-dimensional shape of a compound in the gas phase. Ion mobility separates ions based on their differential mobility through a buffer gas based on the ions shape, charge, and mass. The speed by which the ions traverse the drift region depends on their size: large ions will experience a greater number of collisions and thus travel more slowly than those ions that comprise a smaller cross-section. Thus, ion mobility serves as a useful means of orthogonal separation in unrelated molecules as well. This is a real benefit because the ability to identify and quantify proteins is directly linked to the power of chromatographic separations. 2. Myositis Study. A differential proteomics project comparing the quantitation of proteins from sera of healthy individuals to individuals diagnosed with a rheumatic disease is underway. This work is in collaboration with F. Miller (EAG) as part of the EAGs study of families with twins or siblings discordant for systemic rheumatic disorders. The goal of this project is to determine whether a protein or a suite of proteins can be identified that would allow for the early diagnosis, prognosis, and treatment of these diseases. Sera samples have been depleted and digested, data has been acquired, and the results are being processed. 3. Tissue Study. A protein expression study of LCM tissue is underway. We are collaborating with the LCM group and the Negishi laboratory to identify and quantify proteins observed in liver samples from wild-type and PXR knockout mice (the PXR gene is thought to be involved in the serum and glucocorticoid regulated kinase 2 SGK2). Because of the fact that SGK2 is highly expressed in the pericentral region of liver lobules, it is difficult to decipher the role of SGK2 in gluconeogenesis when using whole liver. To elucidate clear function of SGK2 and SGK2-regulated mechanisms in this region, we are profiling the proteins observed using mass spectrometry. 4. Isoprostane Study. Isoprostanes are a family of compounds that are identical to their corresponding prostaglandins except for differences in stereochemistry. Because isoprostanes can be formed nonenzymatically, they are often studied as markers of oxidative stress. The generation of the most often measured isoprostane, 8-iso-PGF2alpha, by prostaglandin-endoperoxide synthase and arachidonic acid even with low enzymatic activity has been investigated. We recommend using the 8-iso-PGF2alpha/PGF2alpha ratio to quantitatively distinguish between increases in 8-iso-PGF2alpha by enzymatic and/or chemical lipid peroxidation. We have been evaluating plasma sample treatment conditions and chromatographic conditions to achieve the highest recovery and most accurate quantitation of these compounds. Several clinical studies are planned to further characterize the nature of lipid peroxidation in human plasma. 5. Eicosanoid Studies. Eicosanoids and related fatty acid metabolites serve as signaling molecules and are intricately involved in inflammation and cardiovascular health. The level of eicosanoids and eicosanoid metabolites are thought to be involved in many diseases. We are involved in a variety of projects measuring these compounds using mass spectrometry. We use liquid chromatography tandem mass spectrometry to analyze a panel of 49 of these molecules which has allowed us to collaborate with several intramural and extramural researchers. 6. Riluzole Study. Riluzole is a small molecule drug used in the treatment of amyotrophic lateral sclerosis. It may also have use in psychiatric disorders and Alzheimer's disease. Researchers in the Signal Transduction Lab are interested in how biological changes concurrent with the ALS disease state interact with transport and metabolism of riluzole in the brain. Previous studies have quantified plasma levels of riluzole but not brain tissue concentrations. We developed a method for the extraction and quantification of riluzole from rat brain tissue in support of these studies. 7. Steroid Studies. Steroid hormones are widely distributed in nature and are potent signaling molecules. As such, they are of interest to several researchers within the institute. We have done a comparison of the MS sensitivity for a variety of steroids on different instruments in our laboratory. A supported liquid extraction protocol followed by chemical derivatization of estrogens has been developed to allow the simultaneous quantification of androgens, estrogens, and the estrogenic compound diethylstilbestrol in plasma. We are currently evaluating the expansion of the methodology to include corticosteroids and bisphenols. 8. Acrylamide Study. The effects of sterilization parameters on rodent feed have been determined by measuring the amount of acrylamide that is produced in the feed with different sterilization parameters, primarily different steam autoclaving times and number of cycles. Analysis of acrylamide and its genotoxic metabolite glycidamide in rat plasma and urine is currently ongoing. Mass spectral analysis of these low mass polar analytes in complex matrices is challenging. We have developed a novel derivatization method to aid in the extraction, separation, and ionization of these target analytes in biological samples. 9. Clozapine-N-oxide Studies. Over the last decade, a novel chemogenetic tool, DREADDs (designer receptors exclusively activated by designer drugs), has enabled researchers to manipulate cellular activity with increased spatial and temporal specificity. This tool relies on the use of an inert ligand clozapine-N-oxide (CNO), which upon injection can activate or inhibit a mutated receptor. While this method is ideal for studying early neural development in rodents, as manipulation of the embryo may be achieved by CNO injection to the pregnant dams, little is known about the dynamics or mechanisms when CNO is transmitted during this process. Using mass spectrometry, we've developed a method to detect CNO and its metabolites, and increase our understanding of the mechanism of this drug. 10. Bisphenol Studies. Bisphenol A (BPA) has garnered much attention due to its wide distribution in the human environment and potential as an endocrine disruptor. This notoriety has spurred an increase in use of structural analogs of which the biologic fate and activity is less well known. Chloroformate reagents are often used to prepare amino acids and carboxylic acids for gas chromatography based analyses. They are favored for quick reaction in aqueous media at room temperature. Chloroformates also react with phenols, but this ability has been less widely investigated and exploited. In conjunction with the Division of the National Toxicology Program, we have quantified bisphenols (BPA, BPAF, & BPS) in animal feces and intend to expand studies to include organ tissues.